In recent years, the development of alternative energy to oil is a very important issue, because of environmental problems, such as global warming and aerial pollution, in addition to the concern related to transportation energy supply. Plant biomass is the most abundant renewable energy source on earth, which is expected to serve as an alternative source to petroleum. Lignocellulose is the main component of plant biomass, and composed of polysaccharides such as celluloses and hemicelluloses (including xylan, arabinan and mannan), and lignin. These polysaccharides are hydrolyzed into monosaccharides such as glucose and xylose by a variety of glycoside hydrolases, and are used as a biofuel or a raw material of chemical products.
Lignocellulose having a complex structure is persistent and is difficult to degrade or hydrolyze with a single enzyme. For this reason, the hydrolysis of cellulose among the polysaccharides generally requires three types of enzymes: an endoglucanase of glucoside hydrolase (endo-1,4-β-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21). On the other hand, although the structure of hemicellulose may vary depending on the type of plants, for example, xylan is a major constituent in broad-leaved trees, herbaceous plants and the like. For the hydrolysis of xylan, it is thought that xylanase (endo-1,4-(3-xylanase, EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) are required. β-xylosidase is one of the hydrolytic enzymes associated with the process of hydrolyzing the oligosaccharides produced through the hydrolysis of hemicellulose by xylanase to produce monosaccharides.
In the conventional lignocellulose to ethanol conversion process, high-solid loading up to 30-60% in initial substrate concentration has been attempted for the purpose of higher energy efficiency and less water usage. The enzymatic hydrolysis of lignocellulose by such high-solid loading results in the high viscosity of the hydrolyzed biomass solution so that the hydrolysis of lignocellulose hardly proceeds. Therefore, for example, by carrying out the enzymatic hydrolysis process at a high temperature of 80° C. or higher using a thermostable enzyme, in addition to an increase in the hydrolysis reaction rate, since the viscosity of the hydrolyzed biomass solution also reduces, the shortening of the hydrolysis reaction time and the reduction of the amount of enzyme are expected to be achieved. For this reason, for various glycoside hydrolases, development of enzymes that are more excellent in terms of thermostability has been desired.
Many thermostable glycoside hydrolases have been obtained by isolating and identifying the thermophilic microorganisms that live in a high temperature environment, cloning the genes from these cultured and isolated microorganisms and determining the DNA sequence thereof, followed by the expression thereof using Escherichia coli, filamentous fungi and the like. For example, a β-xylosidase derived from filamentous fungi and a β-xylosidase derived from a filamentous fungus Aspergillus oryzae that exhibited an enzyme activity at a temperature of 30° C. have been disclosed in Patent Document 1 and Patent Document 2, respectively. A β-xylosidase derived from Alicyclobacillus acidocaldarius that exhibited an enzymatic activity at a temperature of 50° C. or higher and a pH of 5.5 or less has been disclosed in Patent Document 3. A β-xylosidase derived from Acremonium cellulolyticus that exhibited an enzymatic activity at a temperature of 45° C. has been disclosed in Patent Document 4. In addition, β-xylosidases isolated from certain bacteria and filamentous fungi with optimum temperatures of around 60° C. have been disclosed in Non-Patent Documents 1 to 6.